Search

Celiac disease is triggered by ingestion of gluten from wheat, rye and barley in genetically susceptible patients. In the western population, only 1% of individuals develop celiac disease despite 35% being genetically predisposed. This suggests that environmental exposure plays a role in aetiology of disease. This thesis links bacterial exposure with celiac disease as patients could recognise molecular mimics of gluten derived from Pseudomonas species. We uncovered the mechanism behind immune cell recognition of wheat-gluten peptides and bacterially-derived molecular mimics of these peptides. Future efforts to target key interactions identified in this study may offer therapeutic benefit.

Interleukin-10 (IL-10) is a pleotropic cytokine implicated in infection-associated immunopathology, autoimmunity, and allergy. It was tested as a potential therapeutic agent against several inflammatory and autoimmune disorders. However, systemic IL-10 administration proved to be of limited value, which indicated that its production needed to be carefully targeted in order to be efficacious therapeutically. Hence, research focus shifted on understanding the regulation of IL-10’s downstream targets, so they could be utilised instead in a more cell-specific approach to therapy.Macrophages are regulated by the modulation of metabolic events, and can shift their functional status in response to their changing microenvironmental stimuli. The focus of this project was to investigate how IL-10 impacted on inflammatory macrophage metabolism and whether manipulating targets of IL-10 could result in a reduced inflammatory burden overall. Here, I uncover two targets of IL-10, both part of the arginine-polyamine pathway, as being attractive candidates for modulating macrophage polarisation. The first target, Spermine oxidase (Smox), is a catabolic enzyme of the polyamine pathway, which was upregulated in lipolysaccharide (LPS)-stimulated inflammatory macrophages, but was significantly inhibited in the presence of IL-10. Chemical inhibition of Smox in LPS-treated cells significantly reduced cytotoxicity, pro-inflammatory cytokine secretion, and reactive oxygen species (ROS) production.The second target that is more extensively characterised in this thesis is the mitochondrial isoform of arginase, Arginase-2 (Arg2), which was significantly enhanced by the presence of IL-10 in LPS-stimulated murine and human macrophages. I demonstrated that Arg2 was essential for enhancing mitochondrial respiration in both quiescent and inflammatory macrophage cells. I further delineated that Arg2 enhanced macrophage mitochondrial respiration by enhancing the activity and assembly of a particular node of the electron transport chain at Complex II/Succinate dehydrogenase (Sdh). The product of Sdh, the metabolite fumarate, was found to be downregulated in Arg2–/– macrophages, while the substrate of Sdh, succinate was upregulated. This finding correlated with a significant increase in the expression of the transcription factor Hypoxia Inducible Factor-1α (HIF-1α) and the pro-inflammatory cytokine Interleukin-1β (IL-1β) in the Arg2–/– cells. Collectively, this is the first report of the Arg2 isoform affecting mitochondrial oxidative output in an immune cell type, which in turn modulates the overall inflammatory output of the cell.

Defective haemostasis can contribute to development of a bleeding diathesis, whereas dysregulated coagulation increases the risk of thrombotic events. Acute or chronic inflammation contributes to haemostatic imbalance by promotion of pathological activation of blood coagulation, although the molecular basis for inflammation-driven thrombosis remains poorly defined. In this thesis, I sought to identify novel biological parameters underlying phenotypic heterogeneity in people with severe haemophilia (PWH). Plasma from PWH was found to be more likely to have increased plasma fibrinolytic activity compared to plasma from a healthy age-matched cohort. This was found to arise due to significantly increased plasmin generation activity and/or increased resistance to thrombin-activatable fibrinolysis inhibitor (TAFI) activity in PWH plasma. In addition, a novel positive correlation was identified between increased plasma plasmin generation capacity and development of joint damage in PWH, whereas PWH body weight was inversely correlated with their plasmin generation capacity. In the second part of this study, I showed that macrophage polarisation resulted in divergent effects on macrophage-mediated procoagulant and fibrinolytic activity. Macrophage procoagulant activity induced by exposure to bacterial (lipopolysaccharide, LPS) or viral (single-stranded RNA) mimetics was dependent upon a glycolytic shift in macrophages that could be reversed by chemical inhibitors of glycolysis, thus identifying a novel link between haemostatic macrophage activity and cellular metabolism. Finally, I identified that b-glucan-induced myeloid cell trained immunity in vitro and in vivo lowered the threshold for induction of procoagulant and antifibrinolytic activity by secondary proinflammatory stimuli, and that this phenomenon was dependent upon b-glucan-induced metabolic and epigenetic modifications. Furthermore, I found that the protoporphyrin-IX component of free haem was a novel inducer of trained immunity in myeloid cells, which also promoted exaggerated procoagulant and antifibrinolytic activity upon exposure to subsequent pro-inflammatory stimuli.Collectively, this study used novel assays to define new components of fibrinolytic pathway activity as mediators of phenotypic heterogeneity in PWH. In addition, I identified new molecular mechanisms and cellular pathways underlying myeloid cell procoagulant activity, that may contribute to the pathophysiology of thrombo-inflammatory disease.

Neutrophils are the first cells recruited to an immune stimulus stemming from infection or sterile injuries via a mixture of chemoattractant cues. In addition to eliminating pathogens, neutrophils coordinate the overall inflammation by activating and producing inflammatory signals in the tissue while modulating the activation of other immune cells which in some cases leads to adverse tissue damage. Over amplified or chronic neutrophil recruitment directly leads to autoimmune diseases including rheumatic arthritis, diabetes, neurodegenerative diseases, and cancer. Dampening neutrophil recruitment is a strategy to intervene in neutrophil-orchestrated chronic inflammation. Despite intensive research over the past several decades, clinical studies targeting neutrophil migration have been largely unsuccessful, possibly due to the prominent redundancy of adhesion receptors and chemokines. Additional challenges lie in the balance of dampening detrimental inflammation while preserving immunity. Neutrophils are terminally differentiated cells that are hard to study in cell culture. Mouse models are often used to study hematopoiesis, migration, and chemotaxis of neutrophils but is very labor intensive. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA (miRNA) overexpression screen in zebrafish and identified eight miRNAs as potent suppressors of neutrophil migration. We have generated transgenic zebrafish lines that overexpresses these candidate miRNAs where we recapitulated the mitigation in neutrophil motility and chemotaxis to tissue injury or infection. Among those we further characterized two miRNAs which have not been reported to regulate neutrophil migration, namely miR-722 and miR-199. MiR-722 downregulates the transcript level of rac2 through binding to the rac2 3'UTR. Furthermore, miR-722-overexpressing larvae display improved outcomes in both sterile and bacterial systemic models, which correlates with a robust upregulation of the anti-inflammatory cytokines in the whole larvae and isolated neutrophils. miR-722 protects zebrafish from lethal lipopolysaccharide challenge. In addition, overexpression of mir-722 reduced chemotaxis of human neutrophil like cells, indicating that miR-722 is a potential agent to reduce inflammation in humans. MiR-199, decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (cdk2), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting Cdk2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils without inducing cell death. Human neutrophil-like cells deficient in CDK2 fail to polarize and display altered signaling downstream of the formyl peptide receptor. Chemotaxis of primary human neutrophils is also reduced upon CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. In summary, our results reveal previously unknown functions of these miRNAs, and provide potential avenues to modulate neutrophil migration as well as lead to discoveries of novel factors which can regulate this process. We have also discovered a non-classical role of CDK2 in regulating neutrophil migration which provides directions for alleviating systemic inflammation and a better understanding of neutrophil biology.

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection and is a leading cause of mortality worldwide. The pathophysiology of sepsis is characterised by endothelial dysfunction, hypercoagulation, and sustained hyperinflammation and are key events that lead to multi-organ failure and death. A growing body of literature now suggests that the vascular endothelium plays a critical role in driving early events of sepsis progression. Upon bacterial infection, the endothelium releases a specific class of extracellular vesicle called exosomes. Exosomes are plasma membrane-derived lipid nanovesicles (30 – 120nm) capable of transporting biological material from donor to recipient cells. Biological material including DNA, protein, mRNA, and microRNA (miRNA). While the nature of the signals that drive the sustained hyperinflammatory response observed in sepsis are unknown, there are suggestions that genetic elements may be playing a role. miRNAs are short ~22 nucleotide posttranscriptional and translational repressors of gene expression. They bind mRNA with partial or complete complementarity, which results in translational inhibition of mRNA or its’s catalytic cleavage. Therefore, miRNAs can alter cell signalling pathways, gene expression and physiological responses including inflammation. An increased exosomal load containing a dysregulated miRNome released into the bloodstream by an infected endothelium may be driving the sustained an excessive inflammatory response observed in sepsis. There were three primary objectives of the research presented in this thesis. First, to demonstrate whether Staphylococcus aureus (S. aureus) infection of endothelial cells affected exosome production and exosomal miRNA packaging and expression. Secondly, to deliver S. aureus infected endothelial exosomes to monocytes and observe alterations to the inflammatory phenotype of these cells. Thirdly, to identify the specific miRNA pathways involved in driving the proinflammatory phenotype in monocytes. In this study, we demonstrated that S. aureus infection does not alter exosome production in endothelial cells. However, the small RNA (Delivery of S. aureus infected endothelial exosomes to monocytes increased CD11b and MHCII expression. Both CD11b and MHCII are upregulated in monocytes in response to proinflammatory stimuli and are, therefore, markers of monocyte activation. Increased proinflammatory marker expression correlated with heightened production of the proinflammatory cytokine IL-6 and dampened production of the antinflammatory cytokine IL-10. Serum levels of both IL-6 and IL-10 in sepsis have the highest correlation with mortality ¬in-vivo (3). Interestingly, mTOR protein expression was elevated following delivery of S. aureus infected endothelial exosomes. We demonstrated that inhibition of mTOR using miR-99a and miR-99b mimetics heightened IL-6 and dampened IL-10 production following S. aureus infection. Whereas inhibition of miR-99a and miR-99b using antagomirs facilitated increased mTOR activity which had the opposing effects on IL6 and IL-10 production. Collectively, we have demonstrated that endothelial cells release exosomes containing dysregulated miRNA expression profile following S. aureus infection. These exosomes shift monocytes into a proinflammatory phenotype resulting in heightened IL-6 production, a key proinflammatory cytokine that mediates the initial events leading to sepsis. We demonstrated that this increase in IL-6 production was directly correlated with mTOR activity. Overall, we have identified a novel pathway by which the endothelium drives the proinflammatory phenotype of monocytes in response to infection. Our data suggest that blocking miR-99a and miR-99b inhibition of mTOR following infection may be appropriate treatment strategies for critically ill sepsis patients. Further work is required to assess the effects endothelial exosomes have on other innate immune cells such as macrophages, neutrophils, dendritic cells and to identify the exact mTOR signalling cascade involved.

The human immune system plays a crucial role in health, safeguarding against disease and infection. Central to this protection is the ability of immune cells to distinguish ‘healthy’ and ‘diseased’ cells. However, our understanding of the mechanisms underpinning such recognition is incomplete. My Thesis sought to understand how a class of T cells (γδ T cells) recognise potential infection. Using high-resolution imaging we revealed the molecular mechanisms T cells use to recognise bacterial infection. Complementary work informed how these molecular recognition events may lead to an immune response. These discoveries could pave the way for immunotherapeutic development of such T cells.

Fluorescence microscopy has a resolution limit due to the diffraction of light, which hinders accurate visualisation of nanoscopic organelles and features within the cell. Super-resolution microscopies overcome this limitation using innovative strategies, including single molecule localisations, correlating fluorescence fluctuations, and physical expansion. Each provides a unique advantage for subcellular biology research, such as achieving resolutions as good as 20 nm, the ability to capture live cell dynamics, and decrowd the dense cellular environment. This thesis presents the development of super-resolution microscopy methods dSTORM, SOFI and ExM, and their applications in studying viral protein mechanisms, anticancer drug effects, and genomic organization.

Glucocorticoid-induced leucine zipper (GILZ) is a TSC22 domain family protein induced by GC. This study was designed to examine the post-translational modifications of GILZ and to catalogue its interacting partners, with the aim of gaining a better understanding of how to design a new anti-inflammatory therapy to increase the abundance of GILZ. We investigated the post-translational modifications (PTMs), specifically ubiquitination and phosphorylation of GILZ. The research outcomes from this project may provide new opportunities to target the specific anti-inflammatory actions of GILZ without the devastating side effects of long-term steroid use.

The human immune system consists of hundreds of thousands of nanoscopic molecular machines, referred to as proteins. This thesis explores a group called pore forming proteins, which self-assemble into ring structures in order to insert into and puncture cell barriers. These agents maintain human health by targeting and destroying rogue cells or invading pathogens. If left unchecked, however, the immune system can damage the body. This thesis visualised nanoscopic details of the architecture and organisation of these potent effectors. These findings provide an understanding of the function of these immune weapons and the underlying molecular mechanisms of certain immune diseases.

Neutrophils are significant players in both acute and chronic inflammatory conditions, and function in infectious and autoimmune ailments. MicroRNAs regulate homeostasis in health and disease by fine tuning the expression of a network of genes through post transcriptional regulation. Many microRNAs are expressed in restricted tissues, regulated by physiological conditions such as stress and disease, and are emerging as mediators for intercellular communication that shape the tissue environments. MicroRNA profiles have been recently utilized as biomarkers for diagnosis and prognostic purposes for their stability in plasma and significant correlation with the disease progress. In addition, several microRNAs are in clinical trials for infectious diseases, cardiovascular disorders and cancer. As for neutrophil biology, microRNAs that regulate hematopoiesis and neutrophil development are well known. However, only a few microRNAs are characterized in the context of neutrophil migration and activation by loss of function studies either in mice or in cell culture. In this work we characterize the role of total microRNA regulation on neutrophil function through whole body and neutrophil specific Dicer1 knockout, and identify a microRNA regulator of neutrophil motility. MiR-722 downregulates the transcript level of rac2 through binding to a seed match in the rac2 3'UTR. Furthermore, miR-722 over-expressing larvae display improved outcomes in both sterile and bacterial systemic models. Finally, the miR-722 mimics protect zebrafish from lethal LPS challenge, providing evidence and mechanism of an anti-inflammatory microRNA that restrains detrimental systemic inflammation. We further investigated the role of the inflammatory response in an ischemia-reperfusion model. Extensive future work is required, especially in animal models, to illustrate the pivotal and complex microRNA mediated regulatory network in neutrophils, which is expected to provide the foundation for highly selective microRNA based therapy to control neutrophil behavior in infection and inflammatory disorders.

Neutrophils, contributing to approximately 40%-70% of white blood cells in mammals, are the most abundant type of leukocytes in human circulation. As critical effector cells in innate immunity, neutrophils form the first line of host defense against microbes and are the first immune cells recruited to an inflamed tissue. The pathogen phagocytosis, release of reactive chemicals and proteases, and formation of extracellular traps are the key weapons of neutrophils in host defense. However, neutrophils also contribute to collateral tissue damage when performing their antimicrobial functions. The destructive potential of neutrophils requires the tight regulation of their activation and recruitment. In this study, we found that miR-223 in epithelial cells controls neutrophil response to inflammation through regulating the activation of NF-kB. As fast moving cells, neutrophils rely on glycolysis for energy production. The function of mitochondria in neutrophil motility is unknown. We demonstrated that mitochondria play an indispensable role in neutrophil migration: the biogenesis of mitochondria, mitochondrial ROS and the interaction between mitochondria and ER are all involved in maintaining the movement of neutrophils.